PROJECT SUMMARY This work is designed to understand how proteins encoded by two human deafness genes?CDH23 and PCDH15?assemble to form the mechanosensory apparatus of hair cells in the auditory and vestibular systems. Each hair cell has a bundle of actin-based stereocilia arranged with increasing heights; each stereocilium of a cell extends a filamentous ?tip link? to the next taller stereocilium. Movement of the bundle tightens tip links; they in turn pull open force-gated ion channels that open to depolarize the cell. Thus tip links are at the heart of the inner ear?s function?to turn sound and head movement into neural signals. Each tip link is composed of CDH23 and PCDH15 proteins arranged in an antiparallel hetero-tetrameric filament so as to create two parallel strands with a slight helical twist. This unusual arrangement raises new questions: Why did the tip link evolve to have two strands rather than one? Why is it composed of two cadherin proteins that meet in the middle, rather than one protein that spans the distance between stereocilia? How strong is the bond between cadherins, and how does loud noise break it? Work in the previous project period led to an understanding of the bond at the atomic level, achieved by solving the X-ray crystal structure of the two cadherins where they join. Steered molecular simulations then predicted the mechanism of unbinding and predicted that it would depend steeply on time. But these predictions must be confirmed with biophysical measurements of single-protein unbinding. In this project, we will carry out single-molecule force spectroscopy measurements, to directly measure the unbinding and rebinding of the CDH23-PCDH15 bond. We will first explore the mechanical properties of a single-stranded tip link bond. We will then engineer proteins to contain two CDH23 N-terminal fragments or two PCDH15 N-terminals, to measure the mechanical properties of a double-stranded tip link bond. In each case, we will measure how the unbinding depends on force and on calcium concentration, to understand tip- link rupture with loud sound and in the ionic environment of the inner ear. Finally, we will explore how deafness-causing mutations in CDH23 and PCDH15 compromise the integrity of the tip link. We hypothesize that a double-stranded tip link is far stronger than a single-stranded one for the same reason that trapeze artists hold with two hands: if the cadherins of one strand unbind, they can rapidly rebind because they are held in close proximity, so the overall unbinding rate is greatly slowed. We further hypothesize that hair cells evolved a CDH23-PCDH15 bond that slips by a nanometer or so before release, which in turn makes the unbinding rate force-dependent. A bond that can completely break at very high forces acts as a mechanical circuit breaker, protecting other elements of the mechanotransduction apparatus.